Analyzing Oligos from Biological Matrices

Simplifying the Preparation and Assessment of ADME/Pharmacokinetic Samples

As candidate oligonucleotide therapeutics move through the drug-development path past compound synthesis and in vitro testing, there has been an increasing need to understand how such compounds behave in vivo. ADME/ pharmacokinetics studies require analysis of the chemical structure and quantity of therapeutic oligonucleotides and their metabolites from the time they are introduced into a subject to the point where they are no longer detected in plasma or tissues. These studies are typically performed using liquid chromatography/mass spectrometry (LC/MS).

With small molecule therapeutic analysis, a simple isolation step is usually required to separate the active pharmaceutical ingredient (API) from biological matrices such as plasma or urine. This process is more difficult with oligonucleotides, which are present in small amounts in plasma that is full of proteins, salts, lipids, and cell debris.

These matrix contaminants can interfere with the LC/MS analysis that is used to identify and quantitate the therapeutic oligonucleotide and its metabolites. Further complicating the isolation of oligos from plasma and tissue are their chemical properties. Oligonucleotides’ polar and polyanionic qualities make it difficult to use standard separation procedures to clean up plasma samples for LC/MS analysis methods.

A method for performing sample preparation using a two-step procedure of liquid–liquid extraction (LLE) and solid-phase extraction (SPE) reported in Analytical Chemistry in 2007 has met with limited success with small studies where the large amount of required manual manipulation is not a factor. However, in clinical or animal studies of a candidate therapeutic oligonucleotide, which can require the analysis of thousands of samples, this sample-prep method is not practical.

Improving Oligonucleotide Isolation

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Figure 1. Recovery and extraction effectiveness studies of the isolation protocol using Clarity OTX for a 27 mer phosphorothioate oligonucleotide: The oligonucleotide was spiked into plasma, extracted using Clarity OTX, and analyzed by HPLC. The chromatogram is compared to a control sample run on the HPLC using identical conditions. Near quantitative recovery is observed.

To overcome the limitations of two-step isolation, a new methodology was introduced last year for separating oligonucleotides from serum and plasma. This methodology, which has been incorporated in Phenomenex’ Clarity OTX oligonucleotide isolation kit, uses a novel solubilization buffer instead of the time-consuming LLE steps from other protocols.

This method also uses a mixed-mode SPE sorbent for specifically capturing oligonucleotides while allowing the elution of proteins and lipids that can interfere with LC/MS analysis. Being an SPE-based protocol, the isolation methodology is rapid and easily performed in less than 15 minutes.

The Clarity OTX method uses buffers that maintain near-neutral pH throughout the process to avoid unwanted modifications of the oligonucleotide (depurination for DNA below pH 5 and 2´ to 3´ isomerization for RNA above pH 8). The clean-up and recovery of this protocol is demonstrated in Figure 1.

In this example, a 27 mer DNA oligonucleotide was spiked into plasma at the 12 µg/mL level and isolated using the Clarity OTX protocol for analysis by HPLC. The upper HPLC chromatogram of Figure 1 is the plasma-extracted oligonucleotide; the lower chromatogram is the same oligonucleotide directly injected onto the HPLC system. Note the minimum number of UV-interfering contaminants in the plasma-extracted sample.

Because most matrix interference peaks elute away from the oligonucleotide, quantitation is unaffected by matrix interference. Recoveries of greater than 95% from the plasma samples demonstrate the utility of this protocol for isolating oligonucleotides from biological samples. While the data is not shown here, the new isolation method produces a linear response curve with sensitivity down to low nanomolar concentrations, depending on the LC/MS system used.

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